In early stages of atherosclerosis, macrophages (MF) in the wall of blood vessels encounter lipoprotein deposits that have been extensively modified. These lipoproteins have been acted upon by lipases, causing them to become aggregated, and they are tightly bound to the extracellular matrix. They are also chemically modified (e.g., oxidized). Studies that have used soluble lipoproteins cannot capture essential aspects of the interactions of MF with these retained and aggregated lipoproteins (agLDL) that lead to foam cell formation. Advanced microscopy methods will be used to describe a novel type of interaction in which MF form an extracellular lysosome (a lysosomal synapse), which digests the agLDL. The following aims will be pursued to characterize lysosomal synapse formation by MF: Aim 1. Continue to characterize the novel mechanisms for extracellular hydrolysis of aggregated lipoproteins. A. Determine the molecular mechanisms for lysosomal secretion B. Analyze the mechanisms for transfer of sterol and other metabolites to cells (e.g., role of NPC2) C. Examine the interaction of MF with agLDL in extracellular matrix models that mimic the subintimal space D. Carry out 3D electron microscopy analysis of lysosomal synapses E. Determine whether dendritic cells also form lysosomal synapses with agLDL Aim 2. Analyze the receptors and signal transduction pathways involved in interaction of MF with agLDL A. Examine the role of PI3 Kinase, Akt, and other signaling molecules in the formation and function of the lysosomal synapse using cells from knockout mice, RNAi, and pharmacological agents B. Examine the role of TLR4, Syndecan 4 and other potential surface receptors C. Examine the role of cholesterol and lipid metabolites in activating signaling Aim 3. Examine the formation of lysosomal synapses in mouse atherosclerotic lesions A. Use multiphoton microscopy, injection of fluorescent lipoproteins, and labeled monocytes to determine whether lysosomal synapses can be seen in excised atherosclerotic tissue B. Use rapid biopsies and high pressure freezing followed by 3D electron microscopy to obtain high resolution images of the interaction of MF with agLDL in atherosclerotic lesions. While the importance of immune system cells, especially MFs, in plaque development has been recognized for years, there are many fundamental aspects of the role of these immune system cells that are poorly understood. A major goal is to characterize the signaling events that are triggered by the interaction of MF with agLDL. This may lead to improved understanding of the relationship between atherosclerosis and inflammation. Such insights may help in developing therapies as well as providing deeper understanding of the underlying basis for cardiovascular risk factors such as elevated biomarkers for inflammation.